In the human body, as in all living cells, a paramount function of energy is to synthesize ATP from ADP and Pi. This is accomplished by a ubiquitous and complex enzyme, the ATP synthase. The synthase is embedded in the inner layer of the mitochondrion, whose principal function is to oxidize foodstuffs or synthesis of ATP. With increasing recognition of diseases resulting from mitochondrial dysfunction, it is important to learn how the normal mitochondrion functions. The long-term objective of this research is to gain understanding of the mechanism of ATP synthase. The synthase has 3 copies of catalytic beta subunits and 1,2,3, or more copies of other subunits. A binding change mechanism developed primarily by our laboratory suggests that conformational changes driven by proton translocation across the membrane serve to promote the competent binding of Pi and ADP and the release of tightly bound ADP. The three catalytic subunits in the static enzyme have strikingly different chemical and nucleotide binding properties. During catalysis they are proposed to function in a coordinated sequence so that at any one time each subunit is in a different conformation and positional arrangement with respect to other subunits. One objective of the present research is to find if such positional interchange occurs during catalysis. Methods for radioactive labeling of one subunit in a detectable conformation in the static enzyme are proposed, with the goal of finding if catalytic turnover causes subunit positional interchange. A second objective is to evaluate a new hypothesis that the synthase assumes different forms designed for ATP synthesis or hydrolysis depending on the orientation in the membrane of groups involved in proton translocation. A third objective is to find by use of 18O exchange measurements whether selected enzyme modifiers can uncouple subunit cooperativity and develop new reaction pathways. A final objective is to use 18O exchange and bound nucleotide measurements to learn why Mg2+ markedly inhibits the F1 ATPases and certain anions largely overcome the inhibition.